Fig. 2 | Signal Transduction and Targeted Therapy

Fig. 2

From: Interaction between neutrophil extracellular traps and cardiomyocytes contributes to atrial fibrillation progression

Fig. 2The alternative text for this image may have been generated using AI.

NETs induce the autophagic cell death and mitochondrial injury of cardiomyocytes through the upregulation of autophagy. a Increased p62 positive granules in cardiomyocytes incubated with NETs demonstrated by IF staining microscopy. Cells in 24-well plate on coverslips were stained for DNA (blue) and p62 (red). p62 positive granules were imaged with a confocal laser-scanning microscope. Scale bar: 10 μm. b, c In vitro p62, Beclin-1 and LC3B II/I were all upregulated in cardiomyocytes incubated with NETs (n = 3). d, e In vitro 3-MA partly compromised the upregulation of cleaved- caspase 3 in cardiomyocytes induced by NETs (n = 3). f, g Death rate of cardiomyocytes measured by Celigo analysis in the presence or absence of NETs and NETs+3MA (n = 3). Cells in 96 well plate were stained for all DNA (blue) and nucleuses of dead cells (red). Scale bar: 100 μm. Hoechst single-positive cell was defined as the living, while Hoechst and PI double-positive cell was defined as the dead. h In vitro mitochondrial morphology damage was induced by NETs in cardiomyocytes, including swelling and reduction of cristae. Mitochondria were imaged with transmission electron microscope. Scale bar: 1 μm or 2 μm. i, j In vitro mitochondrial membrane potential was decreased in cardiomyocytes incubated with NETs (n = 5). The mitochondrial membrane potential of cardiomyocytes in 6-well plate in the presence or absence of NETs, degraded NETs with DNase I and supernatants of PMN using JC-1 and Hoechst was imaged by fluorescence microscope. Scale bar: 50 μm. The mitochondrial membrane potential of cardiomyocytes in 96-well plate was measured by fluorescence microplate reader. k, l In vitro excessive mtROS was produced in cardiomyocytes incubated with NETs (n = 3). Cells in 6-well and 96-well plate were stained for DNA (blue) and mtROS (red). mtROS of cardiomyocytes in 6-well plate in the presence or absence of NETs, degraded NETs with DNase I and supernatants of PMN using mitoSOX and Hoechst was imaged by Fluorescence microscope. Scale bar: 50 μm. Cardiomyocytes in 96well plate of increased mtROS production was compared by Celigo analysis. PMN polymorphonuclear leukocytes, mtROS mitochondrial reactive oxygen species. PI propidium iodide. *P < 0.05, **P < 0.01, ***P < 0.001, ns (not significant). Data were presented as mean ± SD

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